Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~3d~
~ETHO~ EQ~ PREPA~I~G R~DUCED ~LVRI~ ~Q~S
Field of the Invention
This invention relates to a method for
S preparing processed foods having reduced calorie
content. More particularly, this invention is directed
to the preparation of a tamarind polysacchariae
hydrolysate and its use as a multi-functional
non-metabolizable food additive. The hydrolysate can be
substituted at high levels not only for metabolizable
carbohydrate components of processed foods, but also for
a portion of fat content without adversely affecting
organoleptic quality.
Backaround and Summary of th~ Invention
Carbohydrates and fats are common constituents
of processed food products. These ingredients have
critical functional significance with regard to the
appearance, taste, mouth feel and other organoleptic
qualities of food. However, fatsJoils and
starch-derived carbohydrates utilized extensively in
processed foods can be metabolized by the human body and
thus contribute significantly to the calorie content of
such foods.
2S In recent years consumers have become increasingly
health consc;ous. Many individuals are attP~pting to
minimize their intake of high-calorie foods and foods
containing high levels of fat. Consumers are demanding
reduced calorie and low-fat versions of traditional
processed foods. Consequently there exists an e~panding
3 !~ r~
need for food additives which can be used as functional
substitutes for the calorie-imparting contents of
processed foods without adversely affecting organoleptic
quality.
I have discovered that a cellulase hydrolysate
of tamarind endosperm polysaccharide meets that need.
The tamarind hydrolysate is unique in that unlike other
carbohydrate hydrolysates it comprises 2 predominant
(typically > 70%) oligosaccharides oligosaccharides
believed to have degrees of polymerization (DP) of 7 and
9, some DP 8 oligosaccharides with most of the remainder
monosaccharides and DP < 6 oligosaccharides. The
hydrolysate can be processed to remove monosaccharides
and DP < 6 oligosaccharides. The tamarind hydrolysate
can be substituted at high levels for a portion of the
metabolizable carbohydrate components of processed foods
without compromising the organoleptic qualities of the
resulting reduced calorie foods. Significantly, its use
also allows reduction of the fat content of those
proce~sed foods.
Tamarind polysaccharide is obtained from the
seed of the tamarind tree, Tamarin~us in~ , a common
forest and cultivated tree found primarily in India,
Burma, Bangladesh and Sri Lanka. Tamarind fruit are in
the form of 10-15 cm long pods consisting of about 55%
pulp, about 34% seed, and about 11% shell and fiber.
Tamarind seed became a commercial ~ource of gum in 1943
when an Indian research institute discovered the gum's
utility as a paper size. Since then, tamarind endosperm
polysaccharide has found many commercial applications.
~ L/~ ~ ,,t 7
In 1988 alone, over 800 metric tons of tamarind seed gum
were e~ported from India.
A variety of uses for the isolated tamarînd
seed polysaccharide have been developed. ~ç~ Rao and
Srivastava, "Tamarind~' in Indu~trial Gums, R.~. Whistler
and J.H. Bemiller, eds., 1973, pp. 402-407. The
polysaccharide has the ability to form jellies with
sugar concentrates over a wide pH range. It has also
been used as a stabilizer in ice creams and mayonnaise.
Further, the textile indus~ry has employed tamarind
polysaccharide for sizing, finishing and printing cotton
and artificial silk. In the cosmetics industry,
tamarind polysaccharide has been used for preparing
emulsions of essential oils, shaving creams and
dentifrices. It has also found use as a binder in the
manufacture of compressed pills and tablets, as an
excipient in making greaseless ointments and as a
gelling agent in the preparation of colloidal iodin~
jelly.
In accordance with the present invention, a
cellulase enzyme hydrolysate of tamarind endosperm
polysaccharide is utilized as a multi-functional, but
non-metabolizable food additive. Further in accordance
with this invention tamarind polysaccharide is converted
in high yield to a food grade hydrolysate believed to
comprise principally, DP 7 and DP 9 oligosaccharides
using commercial cellulase. Preferably the
polysaccharide hydrolysate product is processed, prior
to use in accordance with this invention, to reduce the
amount of oligosaccharides in the hydrolysate having a
~ 3
--4
DP less than 6. In a preferred embodiment th~
hydrolysate is treated to remove at least a portion of
the metabolizable monosaccharides produced during
enzymatic hydroly~is.
Commercially available cellulases selectively
hydrolyze tamarind polysaccharide to produce initially
an oligosaccharide migture comprising DP 7 and DP 9
oligosaccharides. If enzyme action is allowed to
continue, thP DP 9 oligosaccharide is further hydrolyzed
to form a DP i oligosaccharide. Typically, enzymatic
hydrolysis of tamarind polysaccharide in accordance with
this invention is continued until the hydrolysate
solution reaches a near constant viscosity (when DP 7
and DP9 oligosaccharides are the principal
oligosaccharide hydrolysis products), after which time
the hydrolysate solution is heated to terminate
hydrolysis and to precipitate soluble proteins which
then can be removed by filtration. Preferably the
solution is treated to remove at least a portion of the
hydrolysate oligosaccharides of DP less than 6,
optionally decolorized by carbon treatment, and then
freeze dried, spray dried or roll-dried to provide a
multi-functional, yet non-metabolizable food additive as
a free-flowing powder.
The tamarind hydrolysate can be substituted for
up to 60% of the digestible carbohydrates in processed
food products without adversely affecting product
processing or food product organoleptic properties.
Further, it has been found that when the tamarind
polysaccharide is used as a carbohydrate substitute, fat
-) -
content can also be reduced up to 25%. Thus use of the
tamarind hydrolysate as a carbohydrate substitute in
processed foods enables a significant reduction in
calorie content. Significantly, too, because the
hydrolysate has a more homogeneous composition ~a
significant percentage by weight of DP 7-DP g
oligosaccharides) than other art-recognized carbohydrate
hydrolysates, its functional performance is highly
predictable in a wide variety of processPd food
products. Another characteristic of the tamarind
polysaccharide enzymatic hydrolysate which derives from
its predominant DP 7/DP 9 oligosaccharide content is
that, unlike art-recognized food additive gums, the
polysaccharide hydrolysate can be used at high levels in
processed foods without adversely affecting the
processing thereof due to elevated viscosities.
Detailed DescriPtion of the Invention
In accordance with the present invention, there
is provided a process for preparing a food ingredient
comprising DP 7-DP 9 oligosaccharides produced by
cellulase hydrolysis of tamarind polysaccharide. The
product tamarind hydrolysate has been found to exhibit
e~ceptional food-functional characteristics when used as
a substitute for at least a portion of the metabolizable
carbohydrate components of processed foods. Thus in
another embodiment of this invention there is provided a
processed food product having the tamarind hydrolysate
substituted for at least a portion of its normal
metabolizable carbohydrate content. Typically about 1
to about 2 parts of the hydrolysat~ are substituted for
each part of metabolizable carbohydrate deleted from the
processed food recipe.
The non-metabolizable oligosaccharide
composition used in accordance with this invention is
prepared by cellulase hydrolysis of polysaccharides
derived from tamarind seed. Tamarind kernel powder, the
ground endosperm of tamarind seed, or more specifically,
tamarind seed endosperm polysaccharide is converted by
cellulase enzyme action in high yield initially to an
oligosaccharide mixture comprising principally two
oligosaccharides, one having a degree of polymerization
(DP) of 7 and the other having a DP of 9. Upon
prolonged cellulase enzyme action, the DP9
oligosaccharide component is converted to the DP 7
component. The DP 7-DP 9 oligosaccharide products from
cellulase hydrolysis of tamarind endosperm
polysaccharide are, of course, produced in admi~ture
with other oligosaccharides, a small percentage having
DP greater than 9, but most by-product oligosaccharides
having DP less than or equal to 6.
In accordance with a preferred embodiment of
this invention, the enzyme hydrolysate is processed to
remove at least a portion of the oligosaccharides in the
hydrolysate mixture having DP less than or equal to 6.
Such processing can be accomplished, for e~ample, by
membrane filtration (dialysis~ or by art-recognized
chromatographic separation techniques. Most
monosaccharide components of the tamarind endosperm
enzyme hydrolysate are removed by treating the
~ ~3 ~ J~
--7--
.
hydrolysate mixture in solution with active yeast under
conditions condusive to yeast metabolism of the
monosaccharide components of the hydrolysate.
Subsequ~nt to such procPssing to enhance hydrolysate
homogeneity, the hydrolysate composition can be isolated
in solid form by freeze-drying, spray drying or
roll-drying of the hydrolysate solution or by
hydrolysate precipitation techniques.
Tamarind endosperm polysaccharide is
commercially available in a form containing about 8% oil
and in a deoiled form which contains less than about 1%
oil. The deoiled form is preferred for preparation of
the oligosaccharide hydrolysate in accordance with the
present invention. The commercially available
polysaccharide can optionally be purified by dissolution
with heating in water, heating the resulting
solution/suspension at 95C for about 30 minutes,
filtering or centrifuging the solution, and finally
precipitating the purified tamarind polysaccharide from
the filtrate with ethanol.
The exact composition of tamarind endosperm
polysaccharide is not fully known. ~arly work indicated
glucosyl, xylosyl and galactosyl units in the
structure. The ratio of glucosyl:~ylosyl:galactosyl
units in the polysaccharide has been reported as 3:2:1
by a number of workers and as 4:3:1-1.5 by others.
Analysis by methylation and hy~rolysis of the
permethylated polysaccharide suggests a highly branched
chain with non-reducing ends consisting of
D-galactopyranosyl and L-arabinofuranosyl units.
~ ~.9 ~ J 7
--8--
Periodate oxidation of each sugar unit i~dicates the
absence of (1-3) linkages. It has been suggested that
the main chain is cellulose with frequent branching with
short side chains consisting of one or two
D-~ylopyranosyl units capped with D-~ylopyranosyl,
D-galactopyranosyl or L-arahinofuranosyl units.
Conversion of tamarind endosperm polysaccharide
to a hydrolysa~e comprising predominately DP 7-DP9
oligosaccharides is accomplished by action of a
cellulase selected from cellulases of fungal and
bacterial origin. The enzymatic hydrolysis can be
accomplished in aqueous solution containing tamarind
polysaccharide over a wide range of polysaccharide
concentrations. Thus, the reaction can be accomplished
by dissolving tamarind polysaccharide in aqueous
solution at a concentration from about 1% by weight up
to a concentration limited only by polysaccharide
solubility and solution viscosity. Indeed, tamarind
polysaccharide can be added periodically to an ongoing
cellulase hydrolysis reaction mixture to attain
carbohydrate concentrations in the hydrolysate as high
as 50 weight percent.
The cellulase can be selected from any of a
wide variety of commercially available cellulases of
fungal or bacterial origin. Suitable csllulases include
those produced by As~eraillus niaer, Trichoder~ reesei,
pe~nicillium n_tatum, Myrothecium ver~ucaria, ~speraillus
flavus, a~Lqlllus oryzae. Preferred cellulases are
those derived from As~erqillus species and Trichoderma
species. Commercial cellulases from different
sources~organisms have been found to exhihit some
differences in rate of hydrolysis and to some e~tent in
the ratio of oligosaccharides produced by their action
on tamarind polysaccharide. Thus, to optimize
S production of the polysaccharide hydrolysate useful in
accordance with this invention, it is preferable that
each enxyme lot be evaluated in laboratory test runs for
their cellulytic activity on a tamarind polysaccharide
substrate and that such information be used to optimize
conditions for the larger scale production of tamarind
hydrolysate. Commercially available cellulase isolates
from ~LP~~ niq~L (Biocellulase A concentrate, from
Biocon, Inc., Le~ington, Kentucky) and from Trichoderma
reesei (Rohament CT. from Rhom Tech., Inc., Malden,
Massachusetts) have been found to be the most preferred
cellulases for use in production of the oligosaccharide
containing polysaccharide hydrolysates for use in
accordance with this invention.
The amount of enzyme to be used to effect the
requisite hydrolysis of tamarind polysaccharide depends
somewhat on reaction conditions and the activity level
of the cellulase. Under optimum conditions, the enzyme
can be used at levels as low as 0.05 percent weight
relative to the weight of tamarind polysaccharides
starting material. Typically the amount of cellulase
appropriate for production of the hydroysate will range
from about 0.1% to about 5% of the weight of the
tamarind polysaccharide starting material. The time for
accomplishing the hydrolysis reaction likewise will vary
depending on the cellulase hydrolysis conditions.
~ ~3 ~ 7
--10--
:
Typical temperatures range from about 30 to about 50C,
more preferably between about 35 and 45C. U~der
conditions detailed for optimum activity of the
particular cellulase being used, typically detailed by
the enzyme manufacturer in product literature, the
hydrolysis reaction is completed in a period of about 1
to about 12 hours, more typically in about 4 to about 8
hours. The progress of the polysaccharide hydrolysis
reaction can be monitored by standard analytical
techniques such as thin layer chromatography, gel
permeation chromatography or high pressure liquid
chromatography.
The tamarind polysaccharide is hydrolyzed
during cellulase hydrolysis to yield a low viscosity
product comprising, predominantly, two oligosaccharides
of DP 7 and DP9. Continuing the cellulase hydrolysis
reaction results in reduction of the amount of DP9
oligosaccharide and increased amounts of the DP 7
oligosaccharide, presumably by direct cellulytic
hydrolysis of the DP9 oligosaccharide product. Under
typical enzymatic hydrolysis conditions at least 80% by
weight of deoiled tamarind polysaccharides starting
material is converted to tamarind hydrolysate of which
about 70% to about 80~ by weight is a mi~ture of the
DP 7-DP 9 oligosaccharides. The remaining portion of
the crude hydrolysate consists essentially of
monosaccharides and low molecular weight (DP less than
6) oligosaccharides.
The product hydrolysate comprising DP 7/DP9
oligosaccharides is isolated from the hydrolysis
reaction mi~ture by precipitation or solution drying
techniques. Rrotein components in the hydrolysis medium
can be separated, by heating the reaction mixture to a
temperature of at least 90C, preferably between about
9SC and 100C and thereafter filtering the hydrolysate
solution. The heating step effects denaturation and
precipitation of the protein from solution. Remaining
soluble proteins in the filtrate can be removed by
contacting the filtrate with any commercially available
ion e~change resin. This can be accomplished, for
example, either by slurrying the hydrolysate solution
with the resin and filtering or by passing the
hydrolysate solution through a column packed with the
ion-e~change resin.
~nother step for processing the hydrolysate
solution which is desirable but not always necessary is
treatment of the hydrolysate solution with activated
carbon. Such is accomplished by adding activated carbon
(charcoal~, usually in an amount equal to about 5 to
about 20% of the weight of the dissolved hydrolysate, to
the hydrolysate solution, heating the solution and
thereafter filtering, preferably with use of a filter
aid such as celite. Such treatment is effective to
decolorize the hydrolysate solution and to reduce the
concentration of organic impurities.
The crude carbohydrate hydrolysate can be
isolated from the processed hydrolysate solution by
solution drying techniques~ preferab~y lyophilization,
or by precipitation with ethanol. Preferably, however,
the tamarind hydrolysate solution is further processed
~ ~ ~3
12
to remove at least a portion of the monosaccharides and
lower molecular weight oligosaccharides (DP less than
6). Because the principle use of the product tamarind
hydrolysate composition is as a substitute for
S metabolizable carbohydrates in processed foods to reduce
calorie content, it is desirable to minimize the level
of metabolizable monosaccharides in the hydrolysate
composition. Further there is evidence in the
literature that DP2-DP6 oligosaccharides tend to induce
intestinal dysfunction in some people. Thus, it is
preferred that the levels of monosaccharides and DP < 6
oligosaccharides are reduced as much as possible in the
hydrolysate product. This can be accomplished by
membrane filtration or dialysis using commercially
available membranes of controlled pore size.
Monosaccharide content of the crude hydrolysate can be
reduced, as well, by using the crude hydrolysate as a
medium for yeast fermentation. Metabolizable
monosaccharides in the hydrolysate solution are digested
by the growing yeast. Following the yeast digestion
processing step, the yeast cells can be removed from the
hydrolysate solution by centrifugation or other
art-recognized cell-separation techniques. As in the
case of the cellulase hydrolysis of the tamarind
polysaccharide, the progress of yeast digestion can be
followed by standard analytical techniques.
Following the hydrolysate solution processing,
again which can optionally include membrane filtration,
decolorization and/or sterilization, the product
oligosaccharides can be isolated from the hydrolysate
~3~7
solution using standard carbohydrate isolation
techniques such as lyophilization or precipitation.
Selective ethanol precipitation of the hydrolysate
oligosaccharides can be used to provide a hydrolysate
product consistin~ essentially of DP 7-DP9
oligosaccharides without use of the above-mentioned
membrane filtration/yeast processing steps. The higher
molecular weight oligosaccharides, being less soluble,
can be preferentially precipitated leaving more of the
lower molecular weight oligosaccharides (DP < 6) in
solution.
The product oligosaccharide mixture is
typically isolated as a dry, free flowing, white to
cr~am colored powdered. Alternatively, the processed
hydrolysate solution can itself be used as a food
additive as a means for introducing the oligosaccharide
mixture into processed food recipes.
The tamarind polysaccharide hydrolysate can be
used, in accordance with this invention, as a functional
substitute for the metabolizable carbohydrate content of
processed foods to provide reduced-calorie processed
food products. It has been found that a composition
comprising DP 7-DP9 oligosaccharides derived by
cellulase hydrolysis of tamarind polysaccharides can be
substituted for as much as 60% of the metabolizable
carbohydrates in processed food without adversely
affecting organoleptic quality of the modified food
products. Moreover, the use of such oligosaccharide
composition in processed foods allows, as well, a
reduction in fat content without noticeable affect on
-14-
food quality. More particularly, I have ~ound that when
the tamarind hydrolysate (~P 7-DP9 oligosaccharides) is
substituted for about lO to about 40% of the
carbohydrates in a processed food composition, fat
content can be reduced as much as 25%.
The tamarind derived oligosaccharide
composition can be used in accordance with this
invention to produce reduced calorie candy, chewing gum,
dry cake and cookie mixes, frozen dairy desserts,
nutritional bars, gelatin desserts, baked goods and
spoonable dressingsO Further, it may be employed as a
bulking agent without significant increase in
batter~product viscosity. The composition has been
found to dissolve quickly in water ko give clear
solutions. It can be used as a non-caloric carrier for
synthetic sweeteners. When mixed with synthetic
sweetener and added either to ice tea or hot coffee, the
dissolution of the product is instantaneous. It has
been noted as well that the tamarind hydrolysate
oligosaccharide composition can act as a sweetness
intensifier. A baked cookie of high quality can be
prepared by substituting the hydrolysate for about lO to
about 40% of the sugar called for on the original cookie
recipe. In another application of the oli~osaccharide
composition in accordance with the invention, the
composition is combined with dry milk solids to produce
a coffee whitener.
Example 1
Deoiled commercial tamarind seed powder was
sifted into water with vigorous stirring to form a 3% by
~J ~ 3 ~
weight solution/suspension. The solution was heated to
9Q-95C for a 30 minute period with vigorous stirring
and then cooled to 40C before commercial cellulase
enzyme (3% by weight of tamarind powder) from
~ÇLgill~ niger (Biocellulase A Concentrate from
Biocon, Inc.) is added. The reaction was allowed to
proceed with stirring at 40C for about 1~ hours.
The progress of the cellulase hydrolysis
reaction was followed by taking aliquots from the
reaction mixture after 2, 4, 5 and 16 hours,
deactivating the enzyme by heating those samples and
utilizing HPLC analysis to determine the relative
amounts of oligosaccharides present having DP greater
than 9, DP equal to 9, DP equal to 7, DP equal to 1.
The results of such analyses are shown in Table I
below. The data indicate that the hydrolysis reaction
utilizing Biocellulase A converted the tamarind
polysaccharides to the principle DP9 and PD7
oligosaccharide products after 4 hours. Continued
hydrolysis provided further reduction of DP greater than
9 oligosaccharides and DP9 oligosaccharide with
concomitant increase in concentration of the DP 7
oligosaccharide product.
TABLE I
HYDROLYSIS OF DEOILED TAMARIND POWER
(With Biocellulase A Concentrate~
Percent Hydrolysis
ComponentAfter After After After
Present2 hours 4 hours 5 hours 16 hours
DP~916 1 0.4 0.2
30 DP 9 32 25 13 2
DP 745 58 73* 76
DP 1 2 3.6 3 6
~ 1~ 3 ~
-16-
The reaction mi~tur~ was then heatPd to
95-100C for 10 minutes to inactivate the enzyme and to
precipitate at least a portion of the protein present in
the resulting tamarind hydrolysate solution. The
aqueous hydrolysate was then cooled to 60C and filtered
through a Celite pad to remove thP pro~ein precipitate.
The filtrate was tr~ated with activated carbon ~3 6% by
weight of the tamarind powder) to decolorize the
solution and remove organic impurities. ~yophilization
of the decolorized hydrolysate solution provided the
tamarind hydrolysate comprising DP 7 and DP 9
oligosaccharides as a white powder. The viscocity of a
2% aqueous solution of the product was about 1.5
centipoises when measured in an Ostwald Viscometer at
25~C.
E~ample 2
The procedure described in E~ample 1 was
repeated using Rohament CT brand cellulase in place of
Biocellulase A brand cellulase. The reaction was
terminated after seven hours. Table II summarizes the
progress of that reaction after 5 and 7 hours.
TABLE II
HYDROLYSIS OF DEOILED TAMARIND POWDER
(With Rohament CT Cellulase)
Percent Hydrolysis
Component After After
Present_ 5 Hours 7 ~ours
DP>9 7 0.4
DP 9 32 27
DP 7 54 58
DP/l _ 2 _ 2
¢ ~ ~ ~
-17-
The viscosity of a 2~ aqueous solution of the
hydrolysate product is about 2 when measured in an
Ostwald Viscometer at 25C.
E~ample 3
A portion of the dried tamarind hydrolysate
obtained in accordance with Example 1 is dissolved in
water and introduced into a membrane filtration
apparatus utilizing a membrane having a pore size
selected to allow passage of molecules having a
molecular weight of less than 1000. Ethanol
precipitation of the hydrolysate solution a~ter membrane
filtration processing provides a composition consisting
essentially of DP 7 and DP 9 oligosaccharides
substantially free of monosaccharides and
oligosaccharides having a degree of polymerization of
less than 6.
E~ample 4
Following the general procedure described in
Example 1, a 50 gram sample of tamarind endosperm
polysaccharide is hydrolyzed at 40C for 6 hours in 1500
ml of water using 2.0 grams of Biocellulase A
Concentrate (from Biocon, Inc., Lexington, Kentucky)
cellulase enzyme. Using 1.76 gram portions of the
resulting reeze-dried tamarind polysaccharide
~5 hydrolysate dissolved in 10 ml portions of water at pH
5.7, several yeast digestion conditions were evaluated
for effectiveness for reducing monosaccharide content of
the hydrolysate. The hydrolysate solutions were heated
either to 30 or 40 or 50C in a water bath. Yeast
~Fleschmann's Active Dry Yeast), 0.70 grams or 0.106
DI ~ L~. 7
grams, was added and the resulting mi~ture was stirred
for 24 hours or 48 hours a~ 30, 40 or 50. Each
reaction mixture was then boiled 10 minutes to
deactivate the yeast. Insolu~les were removed by adding
Celite to the mi~ture and filtering through a pad of
Celite on Whatman No. 1 filter paper. The
oligosaccharide product was recovered by freeze-drying
the filtrate.
Reduction of monosaccharides by yeast digestion
was quantitated by HPLC analysis. A small amount of
each yeast treated tamarind hydrolysate was passed
through a column of Amberlite 120 cation exchange resin
to remove remaining soluble protein. The filtrate was
concentrated under reduced pressure and chromatographed
on an Aminex HPX87P monosaccharide HPLC column operated
at 60C with a water elutent flow rate of 0.5 ml/min. A
Varian RI detector was used with a Varian 5000 HPLC
pump. The results of the HPLC analysis for the
respective yeast digestions are summarized in Table III.
TABLE III
MONOSACCHARIDE COMPOSITION OF TAMARIND
OLIGOSACCHARIDE MIXTURE (TOM) AND YEAST DIGESTED TOM
Treatment Conditions Monosaccharide Content
Temp. Time Yeast Glc Xyl Gal ~ra
(C) (h~-~ (%) (%~ ~%) ~%) (%)
No treatment 2.4 0.7 1.7 0.8
24 4 0.2 0.7 - 0.4
48 4 0.1 0.8 - -
24 4 0.1 0.8 - 0.2
24 6 0.1 0.8
7 4 - 0.6 1.8 0.8
24 4 0.2 0.7 1.7 0.8
48 4 0.1 0.7
~ ,s3 ~ l~t~
19-
Tamarind polysaccharide hydrolysate produced by
cellulase hydrolysis of tamarind polysaccharide
contained 2.4~ D-glucose, 0.7% xylose, 1.7% D-galactose
and 0.8% L-arabinose as measured by HPLC. Treatment of
the tamarind hydrolysate with 4% by weight of yeast for
24 hours at 30C reduced D-glucose to 0.2%, L-arabinose
to Q.4~, and removed all D-galactose. Continuing the
reaction for 48 hours at 30DC removed the remaining
L-arabinose. HPLC indicated a small peak, 0.1% of the
product, which appeared at the same retention time as
D-glucose. It is believed that the small peak is likely
that of an impurity because the yeast is expected to
completely digest D-glucose. The e~periment indicated
that the most preferred conditions, i.e. those that werP
most effective to reduce monosaccharide content, were
with use of 6% by weight yeast for 29 hours, at 40C.
Yeast digestion under those conditions removes all
monosaccharides except xylose from the tamarind
oligosaccharide mixture.
E~ample 5
Two procedures are presented for enzymatic
hydrolysis of deoiled tamarind polysaccharide to
oligosaccharides with less than 1-2% polysaccharide.
Yield of hydrolysate is about 80% of the tamarind gum.
The hydrolysate contains 70% or more of DP 7 and 9
oligosaccharides, with 9-10% of DP 8, about 10
monosaccharides, and up to 10% of DP 2 to DP 6
oligosaccharides.
Mater;als
Materials used were deoiled tamarind seed
polysaccharide, Biocellulase A Concentrate enzyme, Dowex
-20-
G-60 acti~ated carbon (Aldrich Chemical co.) and Celite
521 (Aldrich Chemical Co.).
General Procedures
A. En~ymatic Hydrolysls Of A 20~ Tamarind
Dispersion ("One~Pot" Process).
Tamarind is sifted into an enzyme and
water solution at 50C with vigorous stirring over 15
minutes in an amount to give a 20% tamarind dispersion
with an amount of Biocellulase A Concentrate enzyme
equal to 1% of tamarind weight. After completion of
tamarind addition, the mixture is stirred an additional
20 minutes at 50C. Vver the next 15 minutes, the
temperature is ~rought from 50C to 70C. Upon reaching
70C, the dispersion becomes viscous, but slow stirring
is continued another 20 minutes at 70C to completely
hydrate the gum. Dispersion temperature is lowered to
50C and additional enzyme equal to 3% of tamarind
weight is added. Hydrolysis at 50C, with stirring, is
continued for 4 hours. To deactivate enzyme after
hydrolysis completion, and to precipitate protein, the
mixture is boiled 10 minutes.
After cooling to near 50CC, the insolubles are
removed by adding Celite to the mi~ture and filtering
through Celite. Filtration is easiest if a small
portion of the mixture is added to the Celite and
allowed to filter through before more is added. Thus,
layers of Celite and insolubles are deposited,
preventing a buildup of slimy proteinaceous material
which greatly slows ~iltration. Celite is washed with
fresh water and filtered again to fully recover
hydrolysate.
~J1~3~L.~7
-21-
Yellow color of tamarind hydrolysate is greatly
reduced with activa~ed carbon decolorization. To the
hydrolysate is added 10% activated carbon, w/w tamarind,
and mixture is stirred at 50C for 2 hours. Carbon is
removed by adding Celite to the mi~ture and filtering
through a thick pad of CeliteO to recover hydrolysate
absorbed on the Celite, the top carbon layer is scraped
off and the Celite washed with fresh water and filtered
through a thin Celite pad.
Yield of hydrolysate (by lyophilization~ is
approximately 80~. The product from a hydrolysis
reaction using 5 lbs of tamarind polysaccharide has a
viscosity of 1 centipoise at 2% by weight in water at
25C.
HPLC ResultS
Hydrolysis was conducted for various time
periods to determine at what point tamarind
polysaccharide was reduced to oligosaccharides. As
shown in Table IV, 4 hours after adding enzyme nearly
all high molecular weight material was converted to
oligosaccharides. Longer hydrolysis times converts DP 9
oligomer into DP 7 and slightly increases monosaccharide
amount. Thus, a hydrolysis time of 4 hours is
recommended.
TABLE IV
HPLC RESU~TS FROM "ONE-POT" HYDROLYSIS
Percent Of Hydrolysat~
3 Hours 4 Hours 5 Hours 6 Hours *
Fraction Hydrolysis ~yd~olysis Hydrolysis Hydrolysis
High MW 5.5 1.1 0.8 0.8
DP 9 30.7 24.1 18.8 15.5
~ DP 7 43.6 46.8 50.0 51.3
Mono-
saccharide 7.4 9.8 10.2 10.9
*Hydrolysis time refers to reaction time after 3% by
weight enzyme addition. Remaining hydrolysate not
included in Table IV is DP 2-~P 6 and DP 8
oligosaccharides.
B. Enzymatic Hydrolysis Of A ~0% Tamarind
Dispersion ~"Two-Pot" Procedure).
Tamarind is sifted into vigorously stirred
water at 25C to give a 25% dispersion. The temperature
is brought to 95C and the dispersion is allowed to
stand 30 minutes at 95C, after which temperature is
reduced to 50C or below. In a separate container,
water equal to one-third the amount used to make the 25%
dispersion is brought to 50C and enzyme equal to 3% of
the tamarind weight is added with slow stirring.
Following dissolution of the enzyme, the thick,
putty-like hydrated tamarind is scooped into the enzyme
solution with vigorous mechanical stirring to assist in
dispersing and breaking up clumps. Addition of all
tamarind requires about 20 minutes and hydrolysis at
50C is continued 4 hours after tamarind is dispersed in
enzyme solution. Following hydrolysis completion,
4; ~ ~ I
-23-
enzyme is denatured and the protein precipitated by
boiling 10 minutes.
Filtration of insolubles with Celite
anddecolorizing with activated carbon is as described
above.
HPLC Results
Enzymatic hydrolysis was conducted for various
time periods to determine length of time necessary to
convert polysaccharide into oligosaccharides,
predominantly DP 9 and 7. As shown in Table V, a
hydrolysis time of 4 hours was required to lower
molecular weight to oligosaccharide level.
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Example 6
Tamarind hydrolysate prepared in accordance
with Example 1 is substituted for portions of the flour
and sugar ingredients in a brownie recipe thereby
reducing the calorie content of the baked product by
about 34~. The modified brownie recipe is as follows:
140 g Granulated sugar
100 g Tamarind hydrolysate
2 g Salt
2.8`g Baking soda
100 g Bread flour
45 g Cellulose fiber
27 g Dutch cocoa
0.5 g Xanthan gum
56 g All purpose shortening
0.4 g Flavoring
48 g Liquid whole eggs
125 g Water
Procedure: Combine granulated sugar and cocoa;
add shortening, and mix. Stir in whole eggs and add
tamarind hydrolysate, salt, baking soda, flour,
cellulose fiber, xanthan gum and mix. Add flour and
water and beat until smooth. Spread batter in a greased
pan and bake at 350F (177C) until sides begin to pull
away from edge of pan.
The flavor and texture of brownies prepared
using the tamarind hydrolysate modified recipe are
comparable in taste, appearance and texture to brownies
prepared utilizing the unmodified brownie recipe.
~33
E~amp.le 7
Tamarind hydrolysate is used to replace about
one-half of the sugar in a lemon-flavored hard candy
recipe reducing the candy's calorie content by about
50%. The modified recipe for this confection is as
follows:
49.0% Tamarind hydrolysate
49.0% Sucrose
0.9% Sodium citrate
0.9% Citric acid
0.15~ Lemon flavor
0.05% F D & C color number 6, lO~ solution
Water (amount sufficient to dissolve
ingredients)
Procedure: Bring water to boil in large, heavy
pan; remove from heat. Add tamarind oligosaccharide and
sucrose and stir until dissolved. Return to heat and
when mixture begins to boil, cover and cook for about -3
minutes. Uncover, and cook at high heat without
stirring until temperature of candy mixture reaches
about 310F (154C). Remove to low heat and stir in
remaining ingredients. Pour candy onto slab or into
molds. Brush with butter or oil.
The reduced calorie candy has a flavor and
mouth feel comparable to candy prepared using the
unmodified recipe.
ExamPle 8
Tamarind hydrolysate is used in the preparation
of a reduced calorie yellow cake as follows:
-27-
2 eggs
1 cup milk
1/2 cup soft shortening
2-1/4 cups sifted cake flour
1 cup granulated sugar
1 cup Tamarind oligosaccharide
3 tsp. double-acting baking powder
1 tsp. salt
1 tsp. vanilla extract
Into large bowl, sift flour, tamarind
oligosaccahride, sugar, baking powder and salt. Drop in
shortening, then pour in 2~3 cup milk and vanilla~ Beat
with electric mixer at medium speed for 2 minutes. Or
beat with spoon in 300 sweeping round-the-bowl strokes,
or 2 minutes by clock, rotating bowl and scraping it
often.
Add 1/3 cup milk and the eggs, unbeaten, and
beat with mixer at medium speed 2 minutes or with spoon
300 strokes - 2 minutes. Pour batter into 2 layer pans,
dividing equally. Bake at 37SF (191C) 25 to 30
minutes, or until they spring back when touched lightly
in center.
The resulting reduced calorie yellow cake is
organoleptically indistinguishable from the unmodified
yellow cake recipe.
ExamPle 9
Tamarind hydrolysate is used as a substitute
for a portion of the sugar in a chocolate pudding mix as
follows:
6.5% Sugar
0.03~ Sodium stearoyl-2-lactylate
- ~J ~ ?3 ~ ~ ~l 7
-28-
3.7~ Tamarind Oligosaccharide
0.47% Sodium pyrophosphate
0.67% Calcium gluconate
0.08% Salt
3.80% Modified starch
1.80% Dutch cocoa
83.2% Skimmed milk
Procedure: Blend together all ingrsdients
except the skimmed milk. Stir in milk and mi~ at low
speed until well blended. Pour into dish and
refrigerate for at least one hour.
The instant pudding prepared in accordance with
the tamarind hydrolysate modii~d recipe has a taste,
viscosity and mouth feel comparable to the pudding
prepared utilizing the unmodified recipe.
Example 10
An artificial sweetener composition is prepared
by blending the following ingredients:
3.5g Calcium saccharin
lOOOg Tamarind hydrolysate
2g Cream of tartar
2g Calcium silicate.
Each 1 gram portion of the resulting free-flowing powder
mixture has the sweetness of two (2~ teaspoons of
25 sugar. It dissolves instantly upon addition to hot or
cold water.
